The subject matter of this disclosure relates to a method of cryopreservation and subsequent thawing of adherent cells attached to a substrate. Also disclosed is a method of identifying one or more extracellular matrix (ECM) components and/or matricellular proteins that improve viability and retention of cells during and after thawing from a cryopreservation state.
The ongoing demand to reduce the number of animals used in research drives the development of in vitro assays, both cell and tissue based, that provide accurate toxicity data about various chemicals, compounds, and formulations. In 2009, the European Union banned the use of animals for testing of cosmetic ingredients. A ban on the use of animals for testing the toxicity of other types of compounds including pharmaceuticals and household chemicals will likely follow.
In response to this demand, cryopreservation of cells and tissues has been used to protect and preserve biological systems by cooling the cells and tissues to temperatures below the freezing point of water. Cryopreservation is generally carried out on cells in suspension and very few studies have examined cryopreservation of cells on a fixed substrate. Accordingly, the commonly used cryopreservation protocols that were developed for cell suspensions are typically applied to adherent cells on a fixed substrate as well, frequently resulting in cell detachment and membrane damage after cryopreservation. Survival of cells from the rigors of freezing and thawing in cryopreservation procedures is only attained by using appropriate cryoprotective agents. Thus, most research in cryobiology has been focused on finding and testing new types of cryoprotectants.
However, retention of cell attachment to the ECM, either natural or synthetic, after the rigors of freezing and thawing is crucial for the preservation of natural and engineered tissues and for other applications such as in vitro toxicology testing. Disruption of adhesive mechanisms inevitably has severe consequences; even reversible effects on the adhesion mechanism can be disastrous because spatial separation from the underlying ECM is sufficient to prevent reattachment.
Accordingly, there is a need for improved cryopreservation protocols for adherent cells. In particular, improved cell attachment and viability after warming the cells attached to a substrate from a cryopreserved state is desired.